The lack of a readily available cell culture system which is permissive for efficient replication of hepatitis C virus (HCV) continues to slow progress in hepatitis C research. The absence of such a system hinders research aimed at characterizing the immune responses to HCV infection, substantially slows vaccine development efforts, and presents a bottleneck to development of effective antiviral therapeutics. This project focuses on the application of molecular approaches to satisfying this critical need, and includes the following specific aims: (1) to develop an infectious cDNA clone of HCV RNA, (2) to force selection of HCV RNA variants with enhanced fitness for replication in cultured cells, using a system involving T7 RNA polymerase-directed in vivo transcription of a large population of viral bicistronic or subgenomic envelope-deletion RNAs which encode a positive selectable marker, (3) to reconstruct infectious cDNA from the selected RNA molecules, and thereby rescue a cell culture-adapted variant of HCV which is capable of efficient replication in cultured cells, (4) to characterize HCV particle formation in vivo in a hybrid eucaryotic T7 expression system, and determine the utility of this system for biochemical and immunologic characterization of HCV particles, and (5) to determine whether variation in the functional activities of regulatory elements within the 5' nontranslated RNA of HCV-infected patients.